We determined anti-HIV-1 activity of GRL-142 and their derivatives against wild-type HIVNL4-3 (HIVWT) and three DRV-resistant HIV-1 variants (HIVDRVR) including HIVDRVRP20, HIVDRVRP30, and HIVDRVRP51. Overall, the nine PIs examined in the present study exerted significantly greater anti-HIV-1 potency (up to 10,000-folds) in cell-based assays and some of them including GRL-002 and GRL-004 showed a much high genetic barrier. The antiviral activity increased when the inhibitors had a cyclopropyl at the P2'-position as compared to an isopropyl together with an aminobenzothiazole moiety. We further investigated the effects of fluorination and replacement of the P2'-Ip-Abo moiety on the cell membrane permeability. The permeability across the cell membrane of PBMCs and MT4 cells for GRL-142 and the nine PIs was significantly higher than that of DRV. Especially, two bis-fluorine- and Abt-containing PIs, GRL-142 and GRL-016, showed 88- and 153-times greater cell permeability than DRV, followed by 4 mono-fluorine- and Abt-containing PIs (GRL-001, GRL-003, GRL-011, and GRL-014). No major difference in cell permeabilities was observed when the fluorine was at the meta- or para-position. We obtained structural insights into the intermolecular interactions between GRL-142 derivatives with PR, based on x-ray crystallography. Crystals of PRWT complexed with three protease inhibitors (GRL-002, GRL-004, and GRL-063) as well as those of GRL-142, GRL-001 and GRL-003 exhibited virtually the identical binding modes, which were found stabilized by a conserved hydrogen bond network. X-ray structural analyses of the PIs complexed with wild-type PR (PRWT) and highly-multi-PI-resistance-associated PRDRVRP51 revealed that the PIs better adapt to structural plasticity in PR with resistance-associated amino acid substitutions by formation of optimal sulfur bond and adaptation of cyclopropyl ring in the S2'-subsite. In order to understand the molecular mechanism of drug resistance, we compared the structure of PRWT with mutant variants PRDRVRP30 and PRDRVRP51; each protein was crystalized in complex with an inhibitor, GRL-001, GRL-003. In the PRDRVRP51 structures, we observed significant structural differences around the 30s loop (residues 29-35). The larger side chain of Phe33 forms stronger hydrophobic interactions inside the hydrophobic cavity that pulls the whole 30s loop towards the segment around Arg20. Consequently, the salt bridge between the residues of Arg-57 and Glu-35 in the PRWT structures was lost in PRDRVRP51, and a new hydrogen bond between Glu-35 and Arg-20 was formed. Concomitantly, the 30s loop lost its contacts with the flap region, which appears to become even more flexible, leading to a widened ligand binding pocket. Particularly, bis-meta fluorine and benzothiazole 2-amino cyclopropyl groups found to be indispensable components to counter activity lost as result of mutations. As a result of optimizing the P1, P2, and P2' positions, the prototypic DRV has been converted to exceedingly potent PIs such as GRL-063 and GRL-016 as well as GRL-142, GRL-001, and GRL-003. They appear to tolerate amino acid substitutions in the PR bindings pocket, making them promising PIs for future clinical development. We also identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C5 position of P2-tetrahydropyrano-tetrahydrofuran (Tp-THF) and P2'-cyclo-(or iso-)propyl-aminobenzothiazole (Abt). Their 50% effective concentrations (EC50s) were 2.3-24 nM against wild-type HIV-1NL4-3 (HIVWT), 0.3-7.9 nM against HIV-2EHO, and 0.9-90 nM against laboratory-selected-PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDRs). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV:HIVDRVRp51), with EC50 values of 38, 62, 61, and 90 nM, respectively, while four PIs examined [amprenavir, atazanavir, lopinavir (LPV), and DRV] virtually had no activity (EC501,000 nM). GRL-078, -079, and -058 structurally form strong hydrogen bond interactions between the C5-modified-Tp-THF and Asp29/Asp30/Gly48 of PRWT. The P2'-Cp-Abt forms strong hydrogen bonds with Asp30'. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. When selected with LPV and DRV using a mixture of 11 HIVMDR strains (HIV11MIX), HIV11MIX became highly resistant to LPV and DRV over 13-32 and 32-41 weeks, respectively. However, when selected with GRL-079 and GRL-058, HIV11MIX failed to replicate at 0.08 microM and 0.2 microM, respectively. Thermal stability and structural analyses help elucidate the highly favorable anti-HIV-1 potency of GRL-079. The present data strongly suggest that the C5-modified P2-Tp-THF and the P2'-Abt moiety contribute to the potent anti-HIV-1 profiles of the four PIs against a wide spectrum of HIVWT and HIVMDRs.